Transcriptional mechanisms underlying sensitization of ...

MOLECULAR PAINBali et al. Molecular Pain 2013, 9:48http://www.molecularpain.com/content/9/1/48

RESEARCH Open Access

Transcriptional mechanisms underlyingsensitization of peripheral sensory neurons byGranulocyte-/Granulocyte-macrophage colonystimulating factorsKiran Kumar Bali1*, Varun Venkataramani1, Venkata P Satagopam2,3, Pooja Gupta1, Reinhard Schneider2,3

and Rohini Kuner1*

Abstract

Background: Cancer-associated pain is a major cause of poor quality of life in cancer patients and is frequentlyresistant to conventional therapy. Recent studies indicate that some hematopoietic growth factors, namelygranulocyte macrophage colony stimulating factor (GMCSF) and granulocyte colony stimulating factor (GCSF), areabundantly released in the tumor microenvironment and play a key role in regulating tumor-nerve interactions andtumor-associated pain by activating receptors on dorsal root ganglion (DRG) neurons. Moreover, thesehematopoietic factors have been highly implicated in postsurgical pain, inflammatory pain and osteoarthritic pain.However, the molecular mechanisms via which G-/GMCSF bring about nociceptive sensitization and elicit pain arenot known.

Results: In order to elucidate G-/GMCSF mediated transcriptional changes in the sensory neurons, we performed acomprehensive, genome-wide analysis of changes in the transcriptome of DRG neurons brought about byexposure to GMCSF or GCSF. We present complete information on regulated genes and validated profiling analysesand report novel regulatory networks and interaction maps revealed by detailed bioinformatics analyses. Amongstthese, we validate calpain 2, matrix metalloproteinase 9 (MMP9) and a RhoGTPase Rac1 as well as Tumor necrosisfactor alpha (TNFα) as transcriptional targets of G-/GMCSF and demonstrate the importance of MMP9 and Rac1 inGMCSF-induced nociceptor sensitization.

Conclusion: With integrative approach of bioinformatics, in vivo pharmacology and behavioral analyses, our resultsnot only indicate that transcriptional control by G-/GMCSF signaling regulates a variety of established painmodulators, but also uncover a large number of novel targets, paving the way for translational analyses in thecontext of pain disorders.

BackgroundPain is one of the most severe and common symptomsof a variety of cancers and is a primary determinant ofthe poor quality of life in cancer patients. In a largenumber of clinical cases, cancer-associated pain, particu-larly the neuropathic component thereof, is resistant to

* Correspondence: [email protected];[email protected] for Pharmacology and Molecular Medicine Partnership Unit,Heidelberg University, Im Neuenheimer Feld 366, D-69120 Heidelberg,GermanyFull list of author information is available at the end of the article

© 2013 Bali et al.; licensee BioMed Central LtdCommons Attribution License (http://creativecreproduction in any medium, provided the or

conventional therapeutics or their application is severelylimited owing to the widespread side effects. Becausemany types of carcinomas and sarcomas metastasize toskeletal bones, they are associated with spontaneouspain, hyperalgesia and allodynia. As potential mecha-nisms, tumor-derived factors, such as NGF [1], endothe-lins [2-4], amongst others, have been studied, whicheither directly activate nociceptive nerves or sensitizethem towards sensory stimuli [5,6].Several types of non-hematopoietic tumors secrete

hematopoietic colony stimulating factors, which act onmyeloid cells and tumor cells [7]. In a recent study, we

. This is an open access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.

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Bali et al. Molecular Pain 2013, 9:48 of 16http://www.molecularpain.com/content/9/1/48

demonstrated that receptors and signaling mediators ofgranulocyte- and granulocyte-macrophage colony stimu-lating factors (G-/GMCSF) are also broadly expressedon sensory nerves in mouse models of bone metastasesas well as in human biopsies of pancreatic adenocarcin-oma [8]. Using animal models of bone metastases whichclosely mimic the nature and progression of cancer painin humans, we reported that GCSF and GMCSF directlyact on receptors on diverse DRG neurons to subserveimportant functions in the generation of pain hypersen-sitivity in tumor-affected regions [8]. Importantly, behav-ioral, electrophysiological and biochemical experimentsdemonstrated sensitization of sensory nerves towardsthermal and mechanical stimuli as well as an increase inneurotransmitter release upon exposure to G-/GMCSF.By adapting RNAi methodology in vivo, we demon-strated that a specific loss of GMCSFRα in DRG led to areduction in bone tumor–evoked pain without inter-fering with the tumor growth, indicating that GMCSFsignaling in peripheral nerves contributes substantiallyto cancer pain [8]. Recent studies on post-surgical painand inflammatory pain also point to a key role for thesecytokines [9-12].G-/GMCSF activates the JAK family of receptor

tyrosine kinases, which unfolds its activity by not onlyregulating enzymes and target proteins within its localmilieu, but importantly also by activating the STAT fam-ily of transcription factors, which subsequently dimerizeand translocate to the cell nucleus to regulate geneexpression [13]. Albeit we have reported local, acuteactivation of the ERK Kinase as well as PI3 Kinase in sen-sory nerves upon a short-term exposure to G-/GMCSF,nothing is known so far about the nature of genes regu-lated transcriptionally in DRG neurons upon exposure toG/GMCSF. However, long-term transcriptional mecha-nisms of G/GMCSF action are arguably of even greaterimportance in pathophysiological states involving chronic,continual release of G/GMCSF, such as tumor-affectedtissues, rheumatoid arthritis, amongst others [14,15].Addressing precise mechanisms via which the G-/GMCSF-JAK-STAT pathway elicits long-term nociceptivesensitization is thus important for understanding mecha-nisms of cancer pain and other chronic disorders asso-ciated with G-/GMCSF release.In lieu of the attractive therapeutic opportunities

offered by these findings, we aimed to elucidate cellulartargets of G/GMCSFR in DRG neurons, particularly withrespect to transcriptional regulation. Not only did wefind a variety of known, established ‘pain-related’ media-tors to be transcriptional targets of G-/GMCSF, but alsoseveral protein-protein interaction hubs were observedto be under G-/GMCSF regulation in sensory neuronsvia detailed bioinformatics analyses. Behavioral andpharmacological analyses on 4 of the emerging targets

confirmed that Rac1 and Matrix metallopeptidase 9(MMP9) contribute to GMCSF-induced nociceptivesensitization. These integrative approaches advance ourunderstanding of chronic pain mechanisms and holdpromise in the development of novel therapeuticapproaches.

Materials and methodsAnimal usageAll animal usage procedures were in accordance withethical guidelines laid down by the International Associ-ation of the Study of Pain and the local governing body(Regierungspräsidium Karlsruhe). All behavioral measure-ments were done in awake, unrestrained, age-matchedadult (more than 2 months-old) C57/Bl6 mice. Mice werehoused in plastic cages, with ambient temperature and a12 h diurnal light cycle. Food and water were providedad libitum.

Sensory neuronal cultures and G-/GMCSF treatmentAdult DRG neuronal cultures were prepared followingthe protocol explained previously [8]. Briefly, neuronalcells isolated from adult wild type mice were seeded onPoly-L-Lysine coated cover slips and maintained in F12Media (Sigma) supplemented with 15% Amino Acids(Gibco), 10% bovine serum (Invitrogen), 1% Penicillin/Streptomycin (Gibco), 0.5% L-Glutamine (Gibco) andNerve Growth Factor (100 mg ml-1, Roche). 4 days oldenriched adult neuronal cultures were starved of growthfactors and serum for 4 h. At the end of 4 h, starvingculture medium was replaced with medium containing0.5% Fetal bovine serum (Life Technologies, 10270106)together with either 1× PBS (vehicle) or 200 ng/mL ofmurine GMCSF (Peprotech, 315–03) or 200 ng/mL ofmurine GCSF (Peprotech 250–05) dissolved in 1×PBS.Neurons were left in the incubator for 24 h. Each treat-ment was performed in triplicate culture wells (n = 3) totest biological variability. At the end of 24 h, total RNAwas isolated and used for microarray expression or qRT-PCR analysis.

RNA isolation from cultured sensory neurons and DRGsTotal RNA from cultured sensory neurons treated withmurine GMCSF or GCSF or PBS for 24 h was isolatedusing mirVana™ miRNA Isolation Kit (Ambion, AM 1561)following manufacturer’s instructions and dissolved in20 μl of nuclease-free water. Purification steps were per-formed using RNAse-free DNAse kit (Qiagen, 79254)following manufacturer’s instructions. RNA concentrationwas determined using the NanoDrop spectrophotometer(NanoDrop Technologies, Wilmington, Germany) and thequality of total RNA was checked by gel analysis using thetotal RNA Nanochip assay on an Agilent 2100 Bioanalyzer(Agilent Technologies GmbH, Waldbronn, Germany).


Only samples with RNA index values greater than 7 wereselected for mRNA profiling. 200 ng of total RNA fromeach biological sample was used as starting material formRNA expression analysis.For in vivo testing, lumbar DRGs L3, L4 and L5 were

collected at 25 h, 36 and 48 h after bilateral intraplantarapplication of 20 ng murine GMCSF and flash frozen inliquid nitrogen. Total RNA was isolated and processedfollowing the same protocol explained above for culturedsensory neurons.

Microarray expression, networking and gene ontologyanalysisThe mRNA profiling was performed on polyadenylatedRNA using Illumina mouse sentrix-6 chips. cDNAlibrary preparation, hybridization and scanning stepswere performed by employing in-house standardizedprotocols and including stringent positive and negativecontrols at each step at the genomics and proteomicscore facility, German Cancer Research Centre (DKFZ,Heidelberg, Germany). The array intensity data wereimported into Beadstudio ver. 3 from Illumina and thequantile array normalization method was employed toaccount for intra- and inter-array variations in expres-sion intensities within each experimental group [16].Magnitude of induction or repression of individual tran-script was compared over vehicle-treated samples. To beable to understand the magnitude of regulation attranscript level, we first converted probe-level signals totranscript-level signals by using in house developed Perlscripts and applying following criteria – i) Take the aver-age fold-change if all the probes for one transcriptshowed the same direction (positive or negative) of regu-lation, ii) Discard the transcripts for which differentprobes showed different directions of regulation iii) Takethe regulation value from the majority of probes if onlyone probe out of several probes is showing differentexpression signals.All the networking analyses of the expression data

were performed using MetaCore™ software in which anetwork is built around an initial list of “seed nodes,”which can originate from the uploaded experiment, orbe manually assembled, or else be automatically conver-ted by MetaCore™ from a list of genes. For the geneontology enrichment analyses, we used software calledbioCompendium (http://biocompendium.embl.de) deve-loped at the European Molecular Biology Laboratory,Heidelberg, Germany.

GMCSF and inhibitors application in vivoMurine GMCSF was purchased from Peprotech, dissol-ved in 1× PBS of physiological PH and 20 ng wasapplied into the intraplantar surface of adult C57/Bl6mice unilaterally for 4 times at 8 h intervals. Inhibitors for

MMP-9 (CAS 1177749-58-4) and Rac1 (1090893-12-1)were purchased from Calbiochem and dissolved in 10%and 50% Dimethyl Sulfoxide (DMSO, Applichem), res-pectively. Calpain 1/2 inhibitor (Calpain inhibitor III,A3672, 0250) was purchased from Sigma-Aldrich anddissolved in 20% DMSO. TNFα inhibitor was purchasedfrom Pfizer (Enbrel®)) and diluted in 1× PBS. One hourafter the last GMCSF dosage application, different groupsof mice received 0.15 pmoles, 1.5 nmoles, 10 nmoles or100 pmoles of MMP-9 or Rac1 or Calpain 1/2 or TNFαinhibitors, respectively, in 10 μl volume of vehicle into thesame paw into which multiple dosages of GMCSF wereapplied. BSA dissolved in 1× PBS was used as vehiclecontrol for TNFα inhibition experiments. Mechanicalhyperalgesia was recorded after 4 and 8 h after the lastGMCSF dosage application while thermal hypersensitivitywas recorded after 5 and 9 h of the last GMCSF dosageapplication.

Mechanical and thermal pain behavioral testsMice were habituated to the experimental setup in atleast 2 separate sessions within the week preceding thetime of behavioral testing. The observer was fullyblinded to the identity of the groups in all behavioraltests. To measure mechanical sensitivity, animals wereplaced on an elevated wire grid and the plantar hindpaw was stimulated using calibrated von Frey monofila-ments of 0.07 g, 0.16 g, 0.4 g and 1.0 g strength (Bioseb,France). Paw withdrawal was recorded as a positiveresponse. Data is expressed as percentage of frequencyof response over 5 stimulations and data from represen-tative filament is shown in this manuscript. For thermalnociceptive testing, radiant heat was applied usingHargreaves’ apparatus (Ugo Basile, Italy) to the plantarsurface of the hind paw, until mice retract it sharply.The time taken to retract (paw withdrawal latency) thehind paw was recorded. A cut-off of 15 seconds heatexposure was followed in order to avoid any potentialdamage to the tissue.

Quantification of mRNA expressionWe used NanoString-nCounter™ based gene quantifi-cation method to validate microarray expression data.Probes specifically targeting the desired gene of inter-est were obtained from Nanostring Technologies,USA and analyses were performed at the nCountercore facility of the Medical Faculty of Heidelberg,Heidelberg University, Germany. Two hundred ng oftotal RNA were used to analyze the expression ofdiverse target genes, using 5 housekeeping genes, namelyClathrin, heavy polypeptide (Cltc), Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), glucuronidase beta(Gusb), Hypoxanthine guanine phosphoribosyl transferase(Hprt) and Tubulin, beta 5 class I (Tubb5), as internal



controls. Expression of target genes was analyzed bycomparing treated and control samples. Fold-change oftest gene was expressed as arithmetic average value overall 5 housekeeping genes.Taqman assays (Life Technologies, USA) were used

for QRTPCR-based quantification of Rac1 (assay IDMm01201653_mH), Calpain2 (assay ID Mm00486669_m1),MMP9 (assay ID Mm00600164_g1) and TNFα (assay IDMm00443260_g1). 20 ng of total RNA was used to preparethe cDNA using random primers from the High CapacitycDNA Reverse Transcription Kit (Applied Biosystems,4368814) following manufacturer’s instructions. Four μl ofprepared cDNA were PCR amplified in each reactionusing mRNA-specific primers and TaqMan® UniversalMaster Mix II, (Applied Biosystems, 4440040) followingmanufacturer’s instructions on Chromo 4 detection sys-tem (BioRad, USA). The expression level of the targetmRNA was normalized to the expression of Glyceralde-hyde 3-phosphate dehydrogenase (GAPDH, assay ID4352932E, Applied biosystems). Each mRNA was ampli-fied from triplicate samples and Ct values were recorded.Fold-change in the mRNA expression in vehicle- orGMCSF-treated sensory neuronal cultures was calculatedusing DDCT method [17] which measures the relativechange in expression of a mRNA from treatment tocontrol compared to the reference gene.

Data analysisAll data are presented as mean ± standard error of themean (S.E.M.), Two-tailed Student’s t-test or the Ana-lysis of Variance (ANOVA) for repeated measuresfollowed by post-hoc Fisher’s LSD test was utilized todetermine statistically significant differences (p < 0.05),unless mentioned otherwise for a particular experiment.

ResultsGMCSF-mediated changes in the gene expressionrepertoire in sensory neuronsTo investigate transcriptional expression changes causedby exposure to GMCSF or GCSF application at agenome-wide level, we performed a genome-wide geneprofiling screen from cultured DRG neurons derivedfrom adult mice. Neuron-enriched cultures were starvedof growth factors and serum for 4 h and treated withGMCSF or GCSF (200 ng/mL in PBS) or vehicle (PBS)in medium containing 0.5% serum for 24 h. Total RNAisolated from 3 such independent experiments wassubjected to quality control as described under methodsand processed in profiling experiments using Illuminaprespotted arrays. cDNA library preparation, hybridi-zation and scanning steps were performed by employingin-house standardized protocols, and including stringentpositive and negative controls at each step. The arrayintensity data were imported into Beadstudio ver. 3 from

Illumina and the quantile array normalization methodwas employed to correct for systematic differencesbetween arrays which do not represent a biologicalvariation of interest between experimental groups [18].Magnitude of fold-induction or fold-repression of indi-vidual transcript was compared over vehicle-treatedsamples, as described in our previous studies [19].Data acquired from the array provided expression sig-

nals from 46090 probes targeting 30723 genome-widetranscripts. Out of 30723 transcripts arrayed 15833 and16882 showed significant fold change in their expressionin GMCSF-treated and GCSF-treated samples, respect-ively, as compared to vehicle-treated samples (P ≤ 0.05,two-tailed t-test assuming equal variance, Benjamini andHochberg false discovery rate correction, n = 3 inde-pendent profiling experiments) (Figure 1 and Additionalfile 1: Table S1). Because both GMCSF and GCSF act ina pronociceptive manner, we then studied commonlyregulated transcripts and found that 3898 transcriptsshowed significant upregulation with GMCSF as well aswith GCSF as stimuli for DRG neurons (Figure 1B).These included several genes which have been im-plicated in nociceptive modulation, such as chemokine(C-C motif ) ligand 2 and 3 (Ccl2 and Ccl3) [20,21],transient receptor potential cation channel, subfamily V,member 1 (TRPV1), a molecular sensor and transducerfor heat, protons and algogens [22], amongst others (afew examples are shown in Figure 1C). Moreover, 9254transcripts were commonly downregulated upon expo-sure with GMCSF as well as with GCSF. These alsoincluded several pain-related known genes, such as thevoltage-dependent calcium channel subunit aplha2/delta1 (Cacna2d1) [23], the AMPA receptor interactingprotein, GRIP1 [24,25], amongst others (Figure 1C). Inter-estingly, however, 421 genes showed reciprocal regulationupon exposure to GMCSF or GCSF (Figure 1B), e.g. thenociceptive modulatory chemokine (C-C motif) ligand(Ccl5) [26] and the matrix metalloprotease 3 (MMP3)[27,28] were significantly upregulated after GMCSFexposure, but downregulated upon exposure to GCSF(Figure 1C). In general, genes encoding nociceptive modu-lating pronociceptive chemokines and cytokines appearedmore strongly regulated by GMCSF signaling than byGCSF signaling in DRG neurons (some examples inFigure 1C).To test the validity of the microarray data reported

above, we performed quantitative measurements of theexpression of several candidate regulated genes usingNanostring-nCounter technique. Amongst putativelyregulated transcripts in the GMCSF-mediated gene pool,we tested 100 genes quantitatively and found that 78genes were regulated as predicted by profiling data,which included up-regulated genes such as chemokine(C-C motif ) ligand 5 (Ccl5), interleukin 1 alpha (Il1a),

Figure 1 GMCSF- or GCSF- mediated gene pool in the peripheral sensory neurons. (A) Heat map representation of significantly-regulatedtranscripts showing more than two-fold change (two-tailed t-test assuming equal variance, P with Benjamini and Hochberg False Discovery Ratecorrection <0.05) following chronic exposure to GMCSF or GCSF in sensory neurons of the DRG. The color scale represents quantile normalizedhybridization intensities for each gene. (B) Venn diagram representing the number of genes commonly or differentially regulated by GMCSFand GCSF exposure in DRG neurons. (C) Selected pain-related genes that were significantly regulated (two-tailed t-test assuming equal variance,P with Benjamini and Hochberg False Discovery Rate correction <0.05). Genes commonly (green text) or differently (red text) regulated by GMCSFand GCSF stimulation in sensory neurons are shown. Gene regulation confirmed by qRT-PCR analysis is highlighted in bold text.


Ccl3 (Figure 2A) and down-regulated genes such asCacna2d1, synapsin II (Syn2), amongst others (Figure 2B).Along the same lines, GCSF-mediated regulation ofseveral genes, including pain-related genes such ascalcitonin/calcitonin-related polypeptide, alpha, transcriptvariant 1(Calca), Ccl3, and fibroblast growth factor 7(Fgf7) amongst several others could be confirmed usingthe same PCR-based methodology (Figure 2C, 2D).These results thus validate the results obtained withthe microarray expression arrays via an independentmethod.

In a next step, to understand systems level interactionsin the GMCSF- or GCSF-mediated gene pools, we perfor-med a direct-interactions analysis using Metacore software[19,29]. When we applied this to all significantly regulatedtranscripts following the criteria explained above forFigure 1, it yielded too dense a network to allow mea-ningful interpretations (data not shown). Therefore, westringently filtered out the transcripts which showed atleast 4-fold up- or down-regulation upon exposure toGMCSF (thus arriving at 661 transcripts) or GCSF (611transcripts). Of these, only 467 GMCSF-target genes and

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Figure 2 Nanostring-nCounter-based validation of selected genes those are up-regulated by GMCSF (A), down-regulated by GMCSF(B), up-regulated by GCSF (C) and down-regulated by GCSF (D) in the sensory neurons. Fold-change expression in the genes is expressedas arithmetic average over 5 housekeeping genes namely Cltc, Gapdh, Gusb, Hprt and Tubb5 in all panels. * P < 0.05, one-way ANOVA followed byFisher’s LSD Post-hoc analysis.


454 GCSF-target genes were well annotated with knownhigher level mapping in Metacore and were used for thedirect-network analysis. The network map generated bythe genego direct-interaction network analysis tool re-vealed a dense network of genes in the GMCSF-targetpool with 3 major nodal points namely, two transcriptionfactors, E26 avian leukemia oncogene 1, 5' domain, tran-script variant 2 (Ets1), Hypoxia inducible factor 1 alphasubunit (Hif1a) and a metallo-protease, namely Mmp9(Figure 3). These 3 nodal points are intensively related tomany kinases such as mitogen-activated protein kinase 3(MAPK3), generic binding proteins such as Synapsin, Rassuper family members such as Rac1, receptors like Toll-like receptor 2 encoding gene (Tlr2), all of which areeither directly or indirectly implicated in nociceptivemechanisms. Similarly, the direct-interaction network forthe GCSF-mediated gene pool also revealed a denselyconnected network with genes encoding the key post-translational sumoylation protein (Sumo1), the cyclin-dependent kinase inhibitor 1A (Cdkn1a), CREB bindingprotein (Crebbp or CBP), calpain 2 (Capn2), MAPK3 andthe RhoGTPase Rac1 (Rac1) serving as major nodalpoints. These nodes are intensively linked to genesencoding Calmodulin 2 (Calm2), the Transient ReceptorFamily channel V1 (Trpv1 or capsaicin receptor), Actin-modulatory protein profilin 1 (Pfn1), among several others

(Figure 4). These results indicate that GMCSF- andGCSF-signaling interlinks transcriptional and post-trans-lational modification mechanisms to key nociceptivemodulatory proteins. We further performed direct inter-action network analysis on genes that were commonlyregulated following GMCSF or GCSF exposure. Thesecommonly-regulated networks revealed shared nodalpoints such as Rac1, mitogen-activated protein kinase 3(Mapk3), among others (Additional file 2: Figure S1 andAdditional file 3: Figure S2).A gene ontology enrichment analysis on the same

subsets of GMCSF- or GCSF-target genes performedusing the bioCompendium software revealed that amajor proportion of transcripts (> 50%) show protein-binding activity. Interestingly, cytokine activity andchemokine-receptor binding categories were found to berepresented in the molecular function enrichment ana-lysis on GMCSF-target pool in DRG neurons (Additionalfile 4: Figure S3), consistent with our observation of highregulation levels of several nociception-related cytokinesand chemokines.In the next step, by using the same subsets of sig-

nificantly regulated GMCSF- or GCSF-modulated genesas explained for Figure 1A, we performed a networkanalysis in which networks are built on the basis of rela-tionships and interactions contained in the MetaCore™

Figure 3 Direct-interactions-network analysis on GMCSF-mediated gene pool with fold-change between +4 and −4 as compared tocontrol-treated sensory neurons and P-BH < 0.05 (t-test, P with Benjamini and Hochberg False Discovery Rate < 0.05). Genes upregulatedand downregulated in GMCSF-dependent manner are marked with red and blue circles, respectively. Please see Additional file 3: Figure S2 forinformation on legends.


Database. Interestingly, the network which emerged witha top rank from the gene pool of GMCSF-targets insensory neurons revealed that the classical signalingcascade consisting of JAK kinases (JAK1 and JAK2) andSTAT transcription factors, STAT1 and STAT3 are tightly

Figure 4 Direct-interactions-network analysis on GCSF-mediated genecontrol-treated sensory neurons and P-BH < 0.05 (t-test, P with Benjamand downregulated in GCSF-dependent manner are marked with red andinformation on legends.

linked to Tumor necrosis factor-alpha (TNF-alpha) andits receptor TNF-R1, both of which were observed to bedirectly regulated by GMCSF in sensory neurons in ourprofiling analyses (Additional file 5: Figure S4). Moreover,a link to NF-kappa-I Kappa B signaling, which has also

pool with fold change between +4 and −4 and as compared toini and Hochberg False Discovery Rate < 0.05). Genes upregulated

blue circles, respectively. Please see Additional file 3: Figure S2 for


been implicated in sensory neurons [30], was also appa-rent (Additional file 5: Figure S4). These findings furtherindicate a close link between GMCSF-induced transcrip-tional control and induction of key nociceptive modula-tors, such as TNF-alpha.

Functional significance of GM-/GCSF-regulated gene poolin GM-/GCSF-induced nociceptive hypersensitivityFinally, to functionally validate our results on GMCSF-and GCSF-associated genes, we selected protein prod-ucts of a set of four candidate genes from differentfunctional classes and with functional relevance to painmodulation, namely the RhoGTPase Rac1, the matrixmetallopeptidase 9 (MMP9), a chemokine TNF-alphaand a generic protease calpain 2. To confirm GMCSF-mediated modulation of these four genes, we comparedtheir mRNA expression in the total RNA isolated fromthe DRG neuronal cultures following chronic treatmentwith GMCSF or vehicle, i.e. a similar paradigm as withthe expression array screening. Analysis of results con-firmed GMCSF-mediated robust upregulation of Rac1,MMP9, TNFα and Calpain 2 as compared to vehicle-treated samples (Figure 5A, *P < 0.05, one-way ANOVAfollowed by Fisher’s LSD Post-hoc analysis).In previous studies, we have analyzed short-term

effects of acute exposure to GCSF and GMCSF [8].

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Figure 5 QRTPCR-based validation of GM-CSF-induced regulation of tas in vivo. Gene regulation at 24 h following the start of GMCSF exposuretime points following intraplantar GMCSF injection is shown in (B). * denotby Fisher’s LSD Post-hoc analysis, n = 3 mice per group. (C) Schematic reprmediated mechanical and thermal hypersensitivity upon inhibition of Rac1

However, in order to mimic chronic clinical conditionswhich are associated with longer exposure to G-/GMCSF and to match the course of the following beha-vioral experiments with the time frame of our generegulation studies, we administered multiple dosages of20 ng murine GMCSF as described in the scheme shownin Figure 5C and inhibitors were applied one hour afterthe last GMCSF dosage application. Mechanical sensitiv-ity was recorded upon ipsilateral plantar application ofgraded von Frey filaments at 3 h and 7 h post injectionand thermal sensitivity was recorded using Hargreaves’apparatus at 4 and 8 h following inhibitor application.For in vivo confirmation of GMCSF-mediated regulationof Rac1, MMP9, Calpain2 and TNFα, DRGs were col-lected at 24, 36 and 48 h after the first GMCSF dosageapplication. QRTPCR-based expression analysis confir-med GMCSF-mediated up-regulation of Rac1, MMP9,Calpain 2 and TNFα in the DRGs of GMCSF-treatedmice as compared to the vehicle-treated group of mice(Figure 5B, *P < 0.05, one-way ANOVA followed by Fisher’sLSD Post-hoc analysis).

Peripheral Rac1 activation is required for GMCSF-mediated mechanical and thermal hyperalgesiaIn our profiling analysis and quantitative PCR analysis,Rac1 expression increased significantly in DRG neurons

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he expression of Rac1, MMP9, Calpain2 and TNFα in vitro as wellin cultured sensory neurons is shown in (A) and in DRGs at differentes P≤ 0.05 as compared to vehicle group, One-Way ANOVA followedesentation of the protocol followed to investigate changes in GMCSF-or MMP9 or Calpain 2 or TNFα in vivo.


following a 24 h-long exposure to GMCSF or GCSF. Inspinal neurons, regulation of Rac1 activity is known toimpact dendritic spine morphology and density as wellas pain hypersensitivity following spinal cord injury [31].However, Rac1 has not been addressed in peripheralsensory neurons in the context of nociceptive modu-lation in sensory neurons. To address whether Rac1 isupregulated at 24 h after exposure to GMCSF contrib-utes to GMCSF-evoked nociceptive hypersensitivity, weselected dosage of the Rac1-specific inhibitor, NSC23766[32], based on the concentration used by Tan et. al [31]in rats. Extrapolating this concentration to mice and toaccount for the dilution factor in the CSF, we selectedapprox. 10 times lesser concentration (1.5 nmoles) inthe current study. We divided mice treated with GMCSFover 24 h into 2 groups – one received a singleintraplantar injection of NSC23766, a specific Rac1inhibitor (1.5 nmoles in 10 μl of 50% DMSO) and theother group received a single intraplantar injection ofvehicle (10 μl of 50% DMSO) 1 h after the last plantartreatment with GMCSF; GMCSF-mediated mechanicaland thermal hypersensitivity was analyzed approx. 3 hand 7 h after inhibitor or vehicle application in bothgroups. Whereas mice injected with vehicle showedsignificant mechanical hypersensitivity to 0.16 g of vonFrey force as compared to vehicle at 3 h as well as 7 hafter the inhibitor application (* P = 0.003 at 3 h and0.007 at 7 h, One-Way ANOVA with repeated measuresfollowed by Fisher’s LSD Post-hoc analysis, n = 6 mice pergroup), mice injected with the Rac1 inhibitor did not showany significant deviation from basal response frequenciesto 0.16 g force (P > 0.05 as compared to basal; n = 6 mice; †P < 0.01 between groups, One-Way ANOVA with repeatedmeasures followed by Fisher’s LSD Post-hoc analysis,Figure 6A and Additional file 6: Table S2). Along the samelines, vehicle-treated mice showed a significant decrease inwithdrawal response latencies to plantar heat (i.e. thermalhyperalgesia) as compared to basal values (* P = 0.003 and0.002 at 4 and 8 h post inhibitor application, respectively,One-way ANOVA followed by Fisher’s LSD Post-hoc ana-lysis, Figure 6B and Additional file 7: Table S3). In con-trast, Rac1-inhibitor-treated mice did not show thermalhyperalgesia at 4 h after inhibitor application; furthermore,thermal hyperalgesia at both time points tested afterinhibitor treatment was significantly reduced as comparedto vehicle-treated mice († P = 0.033 at 4 h and P = 0.019 at8 h between vehicle- and Rac1-inhibitor-treated mice,One-way ANOVA with repeated measures followed byFisher’s LSD Post-hoc analysis, Figure 6B Additional file 7:Table S3). As a control to rule out systemic effects of Rac1inhibitor, we injected inhibitor or vehicle into the pawcontralateral to the paw injected with GMCSF – this treat-ment failed to block GMCSF-induced mechanical andthermal hyperalgesia in the paw ipsilateral to the GMCSF-

injected paw (Additional file 8: Figure S5 panels A and C,*P < 0.05 following GMCSF administration, One-WayANOVA with repeated measures followed by Fisher’s LSDPost-hoc analysis, n = 6 mice). In the same paradigm,mechanical or thermal response frequencies were unal-tered as compared to the basal readings in the paw con-tralateral to the GMCSF-injected paw (Additional file 8:Figure S5 panels B and G, P > 0.05). Similarly, injection ofthe same dosage of Rac1 inhibitor unilaterally into theintraplantar surface in the absence of GMCSF treat-ment did not affect mechanical and thermal responsefrequencies when tested up to 7 h post-injection(Additional file 9: Figure S6, panels A and B). Thus,these results indicate that locally activated Rac1 spe-cifically contributes to both mechanical and thermalhypersensitivity induced by a prolonged peripheral ex-posure to GMCSF.

Peripheral MMP9 activation is required for ongoingnociceptive sensitizationIn the current study, expression of Mmp9, the geneencoding the extracellular matrix protease MMP9,increased by about 5-fold in DRG following GMCSFstimulation, but not following GCSF stimulation. Giventhat MMP9 has been shown to participate in inflamma-tory as well as neuropathic pain in a peripheral as wellas a spinal context [33-35], we were interested inaddressing whether GMCSF-induced upregulation ofMMP9 was functionally linked to GMCSF-evoked exag-geration of mechanical and thermal sensitivity. Using thescheme shown in Figure 5C and described in detailabove for the Rac1-associated experiments, we adminis-tered a single dose of 0.15 pmoles of a potent MMP9inhibitor in 10 μl of 10% DMSO (vehicle) to the plantarsurface 1 h after the last plantar administration ofGMCSF. We selected the MMP9 inhibitor dosage basedon its high potency (IC50 = 5nM) and its reported intra-thecal dosage to attenuate CFA-mediated mechanicalallodynia in rats [36]. Upon peripheral MMP9 inhibition,we observed a complete abrogation of GMCSF-evokedmechanical hypersensitivity to 0.16 g of von Frey forceas well as thermal hyperalgesia at 3–4 h after MMP9inhibitor application (Figure 6C and Additional file 6:Table S2 *P < 0.05 as compared to basal values; † P < 0.05as compared between inhibitor and vehicle groups; One-way ANOVA with repeated measures followed by Fisher’sLSD Post-hoc analysis). This effect on mechanical andthermal hyperalgesia was partially or fully lost, respect-ively, at 7–8 h after inhibitor application, reflecting theduration of action of a single dose of the MMP9-inhibitorat the low dose utilized in this study (Figure 6D andAdditional file 7: Table S3). Similar to the experimentsdescribed above with Rac1 inhibition, we observed thatinjection of the MMP9 inhibitor in the paw contralateral

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Figure 6 In vivo validation of GM-CSF transcriptional targets in DRG neurons in the context of nociceptive sensitization evoked bylong-term exposure to GMCSF. Changes in GMCSF-mediated mechanical hypersensitivity upon inhibition of Rac1 (A), MMP9 (C), Calpain-2 (E)or TNFα (G) as compared to corresponding vehicle-treated mice are shown. Response frequency to the von Frey filament at 0.16 g force isrepresented on the Y-axis. Changes in GM-CSF-mediated thermal hypersensitivity upon inhibition Rac1 (B), MMP9 (D), Calpain-2 (F) or TNFα (H)as compared to corresponding vehicle-treated mice are shown. Withdrawal latency in seconds to calibrated radiant heat is represented on theY-axis. * denotes P≤ 0.05 as compared to basal values, † denotes P≤ 0.05 relative to corresponding vehicle treated group, One-Way ANOVA withrepeated measures followed Fisher’s LSD Post-hoc analysis , n = 6 mice per group.


to the paw injected with GMCSF did not significantlyinfluence GMCSF-mediated mechanical and thermalhyperalgesia in the paw ipsilateral to the GMCSF-injectedpaw (Additional file 8: Figure S5 panels B and D, *P < 0.05following GM-CSF administration as compared to basalvalues, One-way ANOVA with repeated measures fol-lowed by Fisher’s LSD Post-hoc analysis) nor inducedhyperalgesia in the paw contralateral to the GMCSF-injected paw (Additional file 8: Figure S5, panels F and H,

P > 0.05). Furthermore, injection of the MMP9 inhibitor inthe absence of GM-CSF did not affect nociceptive sensi-tivity (Additional file 9: Figure S6 panels C and D, P > 0.05as compared to basal values, One-way ANOVA withrepeated measures followed by Fisher’s LSD Post-hocanalysis). These results indicate that similar to Rac1, peri-pheral MMP9 activation is important for ongoing noci-ceptive sensitization that develops upon a prolongedexposure to GMCSF.


Peripheral calpain2 and TNFα are dispensable in themediation of GMCSF-mediated hyperalgesiaIn the current study, Capn2, which encodes the calcium-dependent cysteine proteases calpain 2, emerged as a geneshowing a high level of induction, common to bothGMCSF and GCSF, in sensory neurons. Drugs whichinhibit both Calpain 1 and Calpain 2 have been shown toinhibit mechanical hyperalgesia following inflammation orin response to some mediators [37,38]. Therefore, we alsotested the potential of a peripherally administered Calpain1/2 inhibitor (Calpain inhibitor III, 10 nmol in 10 μl of10% DMSO) to modulate GMCSF-mediated mechanicaland thermal hyperalgesia using the paradigm describedabove and inhibitor concentrations that have been shownto be effective in previous studies on inflammatory pain[37,38]. Our analysis showed, however, that GMCSF-induced hypersensitivity was not significantly differentbetween mice receiving Calpain1/2 inhibitor or vehicle(10 μl of 10% DMSO) in all tests and at all time pointstested (Figure 6E and F; Additional file 6: Table S2 andAdditional file 7: Table S3; * P < 0.05 as compared to basalvalues; One-way ANOVA with repeated measures follo-wed by Fisher’s LSD Post-hoc analysis).In the current study, tumor necrosis factor alpha (TNFα)

was upregulated by 10-fold following GMCSF stimulationbut only moderately following GCSF stimulation in micro-array or QRTPCR experiments. TNFα inhibition in thespinal cord has been reported to be protective againstneuropathic pain [39-41] and systemically-applied TNFαinhibitor protects mice from tumor-induced thermalhyperalgesia [42]. Moreover, a specific TNFα inhibitor,namely Etanercept (trade name Enbrel®), has been sug-gested to be efficacious against autoimmune diseases suchas Crohn’s disease [43] and is in clinical practice for thetreatment of several peripheral inflammatory diseases suchas rheumatoid arthritis [44]. Therefore, we sought out toinvestigate the potential of peripherally applied TNFα decoyreceptor, Etanercept, choosing a dose of 100 pmol, which isslightly higher than the dose reported to reduce neuro-pathic hypersensitivity [40]. We observed that GMCSF-induced mechanical and thermal hyperalgesia were notsignificantly different between groups treated with vehicle(BSA) or Etanercept (Figure 6G and H, Additional file 6:Table S2 and Additional file 7: Table S3; * P < 0.05 followingGM-CSF administration as compared to basal values, One-way ANOVA with repeated measures followed by Fisher’sLSD Post-hoc analysis). Thus, albeit TNFα is upregulatedin peripheral sensory neurons following GM-CSF admin-istration, it does not appear to directly contribute to GM-CSF-induced nociceptive hypersensitivity.

DiscussionWe and others have previously demonstrated that thehematopoietic growth factors, GMCSF and GCSF, sensitize

sensory nerves directly via activation of receptors locatedon sensory nerves and contribute significantly to cancer-associated pain [8]. Several recent studies have extendedthese results to acute and chronic pain in the context ofpost-operative pain, inflammatory pain and osteoarthriticpain [9-12]. Thus, understanding the genomic programinduced in sensory neurons by this set of key cytokines iscrucial for gaining insights into the pathophysiological roleof G-/GMCSF, as well as in developing therapeutic options.To understand the molecular mediators via which theGMCSF receptor is exerting its protective pronociceptiveaffects, we performed a genome-wide expression screen incultured sensory neurons following G-/GMCSF ligandtreatments. Using in-depth systems level in silico analysis,and in vivo pharmacology to functionally test selectedcandidate genes, we present G-/GMCSF-mediated tran-scriptome change and its importance in modulatingGMCSF-mediated hyperalgesia in the sensory neurons.One of the most interesting observations of the

present study was that many chemokines such as Ccl3,Ccl5, insulin-like growth factor 1 (Igf1), Vascular endo-thelial growth factor A (Vegfa), transcript variant 2 andTNFα, which are known to play a significant role in painmodulation [20,26,45,46] are regulated in a G-/GMCSFdependent manner in sensory neurons. It was previouslyreported that the lack of CCL5 results in decreasedhypersensitivity in partial sciatic nerve ligation model ofneuropathic pain. Further, infiltration of pro-inflammatorycytokines such as tumor necrosis factor-α, [is this supposedto be alpha?] interleukin [IL]-1b, IL-6, and interferon-c wassignificantly reduced in the damaged nerves while that ofanti-inflammatory cytokines such as IL-4 and IL-10 wassignificantly increased in the injured nerves following theglobal CCL5 loss in mice [26]. TNFα was shown to have asignificant contribution in the development of pain sensi-tivity following peripheral nerve injury.Rac1 is a small GTPase ubiquitously expressed in

neurons and other cell types. Spinal cord neurons, astro-cytes and oligodendrocytes express high levels of Rac1mRNA and its expression is elevated following spinal cordinjury (SCI) in rats [47]. Intrathecally applied specific Rac1inhibitor ameliorates SCI-induced changes in spinemorphology, attenuates injury-induced hyper excitabilityof wide-dynamic range neurons, and increases SCI-mediated pain thresholds [31]. Rac1 activity regulates den-dritic spine morphology in hippocampal neurons throughactin cytoskeleton reorganization and promotes clusteringof glutamate AMPA receptors in spines [48-50]. In thecurrent study, Rac1 expression increased by about 4–5fold in sensory neurons following exposure to GMCSF orGCSF stimulation, suggesting that this may work as ashared mechanism between GMCSF and GCSF stimuli tomodulate functional as well as structural changes insensory neurons. Indeed, we have previously shown that


knocking down GMCSF receptor-alpha in sensory neu-rons of the DRG attenuates tumor-induced structural re-modeling and sprouting of peripheral sensory terminals[8]. Further, inhibiting Rac1 activity in the peripheral ter-minals resulted in complete protection from GMCSF-induced mechanical hypersensitivity and partial protectionfrom thermal hypersensitivity. These effects of peripher-ally applied Rac1 inhibitor were devoid of a systemic com-ponent and did not come about via an alteration of basalmechanical and thermal sensitivity. These results under-line the importance of Rac1 in the periphery in the modu-lation of nociceptive stimuli, in the context of bothmechanical and thermal modalities.MMP9 is under the transcriptional control of Ets1 and

belongs to a family of extracellular proteases calledmatrix metalloproteinases (MMPs) [51]. MMPs play acritical role in neuroinflammation through the cleavageof the extracellular matrix (ECM) proteins, cytokines,and chemokines [52-54]. Consistent with previouslyreported changes in MMP9 expression in nervous tissuefollowing damage to peripheral nerves [33,35,52,55,56],we observed a 5-fold increase in MMP9 expressionfollowing GMCSF stimulus in sensory neurons. Intra-thecally administered MMP9 inhibitors have been shownto block mechanical allodynia associated with peripheralinflammation [36] as well as nerve injury [35]. Func-tional experiments performed in the current study showthat MMP9 inhibition in the periphery leads to significantprotection from GMCSF-mediated mechanical hypersen-sitivity in the absence of a systemic influence or withoutaffecting basal nociception. Interestingly, these findingsthus indicate that MMP9 exerts pronociceptive effects notonly in the central terminals but also at the peripheralnerve endings. Our findings further suggest that MMP9may be linked to the role of GMCSF in tumor-associatedpain, since both inflammatory and neuropathic mecha-nisms contribute to cancer pain. However, peripheralblockade of MMP9 did not affect GMCSF-mediated ther-mal hyperalgesia, which is consistent with a lack of its rolein inflammatory thermal hyperalgesia.Calpains are a family of ubiquitously expressed

calcium-dependent cysteine proteases and have beenimplicated in several cellular processes such as prolifera-tion, apoptosis and differentiation [57-59], pathologicalconditions including neuronal plasticity [60], neuronalcell death [61]. Proteolytic targets of calpain includecytoskeletal proteins such as neurofilament, spectrin,and membrane proteins such as growth factor receptors[62,63], cytokines [64,65] and transcription factors [66].In the current study, long-term GMCSF or GCSF ex-posure led to robust transcriptional increase in the ex-pression of calpain 2 and 7, but not other calpain familymembers, in sensory neurons. However, inhibiting thecalpain 2 protease by using a specific calpain I/II inhibitor

in the periphery did not have any impact on modulatingGMCSF-induced mechanical and thermal hyperalgesia.This lack of phenotypic changes is not due to a lack ofefficacy of the inhibitor since intrathecal application ofcomparable concentrations has been shown to signifi-cantly reduce zymosan-mediated thermal hypersensitivityin rats [37] or lysophosphatidic acid (LPA)-mediated ther-mal hyperalgesia in mice [38]. Thus, our results indicatethat calpain is dispensable for GMCSF-induced nocicep-tive sensitization in the periphery; however, they do notrule out that GMCSF-mediated induction of calpain ex-pression may be modulating other functions and processesin the DRG which were not studied here.TNFα is a proinflammatory chemokine which was previ-

ously studied intensively in the context of nociceptivemodulation [67,68]. Importantly, intraperitoneal applica-tion of TNFα decoy receptor etanercept relieved mice fromtumor-mediated hyperalgesia [42]. However, it was alsoreported that intrathecally and intraperitoneally appliedetanercept protects the mice from diabetic neuropathy-induced mechanical hyperalgesia but intraplantar applica-tion of the same inhibitor showed inefficient in protectingthe mice from diabetic neuropathy-induced hypersensitiv-ity [40]. Consistent with this observation, we observed thatperipheral inhibition of TNFα is not sufficient to abrogatethe nociceptive stimulus-mediated sensitivity. Taken to-gether, these observations suggest that TNFα is recruiteddownstream of other tumor-associated mediators.Other than the genes directly regulated by G-/GMCSF

transcriptionally, our systems level network analysis re-vealed several other pathways which could be directlyconnected to the G-/GMCSF-mediated genes, such as IKK-NF-κB pathway. This classical, canonical pathway involvesTNFα or IL-1β stimuli via respective receptors leading tothe activation of an inhibitor of the NF-κB (I-κB) kin-ase complex, consisting of the regulatory subunit I-κBkinases (IKKs). This pathway is crucial for the activa-tion of innate immunity and inflammation [69]. Function-ally, increased NF-κB levels in DRG neurons followingsciatic nerve crush [70] or upregulation of NF- κB transac-tivation following sciatic nerve transection [71] have beenreported. Pharmacological intervention at several nodes ofthe NF-κB pathway has been shown to modulate nocicep-tive responses [69]. Inactivation of NF-κB specifically inprimary sensory neurons of DRG via Cre-LoxP-mediateddeletion of IKKB inhibitor kappa B kinase beta (IKKβ) hasbeen shown to have a protective effect on nerve injury-mediated hyperalgesia [72]. Interestingly, the expression ofTNFα and IL-1β robustly increased following G-/GMCSFstimuli in the current study, implying a clearer activationof NF-κB pathway in sensory neurons. Another molecularpathway which emerged to be very closely connected toG-/GMCSF-mediated transcriptome in sensory neurons iscaspase signaling. Caspases are a family of proteases and


play an important role in mediating programmed celldeath following different noxious stimuli [73,74]. Periph-eral nerve injury promotes neuronal cell death in thespinal dorsal horn [75-77] and arresting this nerve injury-induced neuronal loss in the spinal dorsal horn byblocking caspase activity reduces neuropathy-induced painsensitivity [78]. Caspase signaling pathways differentiallycontribute to neuropathy-induced and TNF-mediatedpain behaviors [79]. Our results on network analysis indi-cate that GMCSF signaling may be interlinked with TNF-alpha and caspase signaling in DRG neurons.Thus, the striking changes we report in the transcription

of many pain-related ion channels, chemokines, growthfactors and proteases among several other classes of genesin DRG neurons following prolonged exposure to G-/GMCSF imply that G-/GMCSF signaling is a trigger pointfor activation of multiple pain modulatory pathways andthat blocking the G-/GMCSF signaling may be very effect-ive in alleviating a broad set of pain disorders.

ConclusionIn summary, the current study demonstrates genome-wide transcriptome changes following chronic G-/GMCSFstimulus in the sensory neurons. Utilizing state-of-the artin silico systems level analysis, this study not only revealsthat several key pain-related genes to be transcriptionaltargets of G-/GMCSF signaling, but also provides novelinsights into network interactions with several other novelcandidate genes. Using in vivo pharmacology, we providethe importance of peripheral MMP9 and Rac1 signaling ininhibiting GMCSF-mediated mechanical and thermalhypersensitivity. Thus, with integrative approach of gen-omics, bioinformatics, in vivo pharmacology and behav-ioral analyses, this study advances the understanding ofnociceptive mechanisms in sensory neurons and providesa basis for further pursuing G-/GMCSF signaling in thera-peutic treatment of pain disorders.

Additional files

Additional file 1: Table S1. Significantly regulated genes followingGMCSF or GCSF chronic stimulus in the sensory neurons with the 2 foldcut-off and pBH < 0.05 (two-tailed t-test assuming equal variance, P withBenjamini and Hochberg False Discovery Rate correction <0.05).

Additional file 2: Figure S1. Symbols used to represent differentfunctional classes of protein to represent direct-interactions networks inFigures 3 and 4 and network interactions in Additional file 3: Figure S2and Additional file 4: Figure S3.

Additional file 3: Figure S2. Direct-interactions-network analysis on thegene pool commonly up- and down-regulated following GMCSF or GCSFexposure in the sensory neurons. Gene pool with fold-change between +4and −4 as compared to control-treated sensory neurons and P-BH < 0.05(t-test, P with Benjamini and Hochberg False Discovery Rate < 0.05). Genesupregulated and downregulated in GMCSF-dependent manner are markedwith red and blue circles respectively. Please see Additional file 2: Figure S1for information on legends.

Additional file 4: Figure S3. Gene-ontology enrichment analysis ongene pools induced by GMCSF (A) and GCSF (B). Analysis was performedusing bioCompendium online repository (http://biocompendium.embl.de) using the gene pool with fold-change between +2 and −2, ascompared to control-treated sensory neurons and P-BH < 0.05 (t-test, Pwith Benjamini and Hochberg False Discovery Rate <0.05). Percent of G-/GM-CSF induced genes over total input number of genes significantlyenriched are represented in both panels (P-BH < 0.05, as compared tomouse genome, hypergeometric statistical method).

Additional file 5: Figure S4. Top scored network obtained from thegene pool regulated by GM-CSF stimulus in sensory neurons, using thesame set of genes explained in the Figure 1-A. Genes upregulated anddownregulated in GMCSF-dependent manner are shown with red andblue circles respectively. Thick cyan lines indicate the fragments ofcanonical pathways. Please refer Additional file 2: Figure S1 for thesymbols representing different generic classes of proteins.

Additional file 6: Table S2. Impact of inhibition of Rac1, MMP9, Calpain2or TNFα on GMCSF-mediated mechanical hypersensitivity. Responsefrequencies to calibrated von Frey filaments of different strength at pawsipsilateral and contralateral to intraplantar GMCSF administration are shownas compared to corresponding vehicle-treated mice. * denotes P≤ 0.05 ascompared to basal values, † denotes P≤ 0.05 relative to correspondingvehicle-treated group, One-Way ANOVA with repeated measures followedFisher’s LSD Post-hoc analysis , n = 6 mice per group.

Additional file 7: Table S3. Impact of Inhibition of Rac1, MMP9,Calpain2 or TNFα on GMCSF-mediated thermal hypersensitivity.Withdrawal latency in seconds to calibrated radiant heat from the pawsipsilateral and contralateral to intraplantar GMCSF administration areshown as compared to corresponding vehicle-treated mice. * denotesP≤ 0.05 as compared to basal values, † denotes P≤ 0.05 relative tocorresponding vehicle treated group, One-Way ANOVA with repeatedmeasures followed Fisher’s LSD Post-hoc analysis, n = 6 mice per group.

Additional file 8: Figure S5. Systemic effects of intraplantar applicationof inhibitors of specific pathways on GMCSF-induced nociceptivesensitization. Changes in the GMCSF-mediated mechanicalhypersensitivity in the paw ipsilateral to the GMCSF-injected pawfollowing Rac1 (A) or MMP9 (B) inhibitors application in the pawcontralaeral to GMCSF-injected paw or changes in the GM-CSF-mediatedmechanical hypersensitivity in the paw contralateral to the GMCSF-injectedpaw following Rac1 (E) or MMP9 (F) inhibitors application in the pawcontralaeral to GMCSF-injected paw as compared to corresponding vehicle-treated mice are shown. Response frequency to the von Frey filament at0.16 g force is represented on the Y-axis. Changes in the GMCSF-mediatedthermal hypersensitivity in the paw ipsilateral to the GMCSF-injected pawfollowing Rac1 (C) or MMP9 (D) inhibitors or changes in the GMCSF-mediated thermal hypersensitivity in the paw contralateral to the GMCSF-injected paw following Rac1 (G) or MMP9 (H) inhibitors as compared tocorresponding vehicle-treated group of mice. Withdrawal latency inseconds to calibrated radiant heat is represented. * denotes P≤ 0.05 ascompared to basal values, One-Way ANOVA with repeated measuresfollowed Fisher’s LSD Post-hoc analysis, n = 6 mice per group.

Additional file 9: Figure S6. Effect of blockade of Rac1 or MMP9 on basalmechanical and thermal sensitivity. Changes in the perception of mechanicalsensitivity in response to von Frey filaments of increasing strength in the pawipsilateral to the Rac1 (A) or MMP9 (C) inhibitor application is compared tothe response frequency before inhibitor application (basal). PercentageResponse frequency to the von Frey filament at 0.16 g force is representedon the Y-axis. Changes in the response to thermal stimuli following Rac1 (B)or MMP9 (D) inhibitors as compared to basal reading. Withdrawal latency inseconds to calibrated radiant heat is represented. * denotes P≤ 0.05 ascompared to basal values, One-Way ANOVA with repeated measuresfollowed Fisher’s LSD Post-hoc analysis , n = 6 mice per group.

AbbreviationsDRG: Dorsal root ganglion; GMCSF: Granulocyte macrophage colonystimulating factor; GCSF: Granulocyte colony stimulating factor;PBS: Phosphate buffered saline.

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Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsKB performed the major portion of the experiments, analyzed data andwrote the manuscript. VV and PG contributed to experiments and performeddata analysis. VPS and RS contributed to the bioinformatics part of the study.RK designed and supervised the study and wrote the manuscript. All authorsread and approved the final manuscript.

AcknowledgementsThe authors are grateful to Rose LeFaucheur for secretarial work and toDunja Baumgartl-Ahlert for technical assistance. This work was supported bygrants from the Association for International Cancer Research and theSander Foundation to RK. RK is a principal investigator in the ExcellenceCluster ‘CellNetworks’ of Heidelberg University.

Author details1Institute for Pharmacology and Molecular Medicine Partnership Unit,Heidelberg University, Im Neuenheimer Feld 366, D-69120 Heidelberg,Germany. 2Luxembourg Centre for Systems Biomedicine (LCSB), University ofLuxembourg, Campus Belval, House of Biomedicine, 7 avenue des Hauts-Fourneaux, L-4362 Esch-sur-Alzette, Luxembourg. 3European MolecularBiology Laboratory, Meyerhofstrasse. 1, D-69117 Heidelberg, Germany.

Received: 21 March 2013 Accepted: 25 August 2013Published: 25 September 2013

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doi:10.1186/1744-8069-9-48Cite this article as: Bali et al.: Transcriptional mechanisms underlyingsensitization of peripheral sensory neurons by Granulocyte-/Granulocyte-macrophage colony stimulating factors. Molecular Pain2013 9:48.

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